Hi everyone,
Recently, I have been working on effect of a protein on the expression or activity or stability of another protein. But, I know one thing that the protein which is modulating the expression or activity of the other protein, is actually doing by controlling a cellular pathway. So, my question is:
If A controls the half-life/or post-translational stability of B. Then, possibly how can I determine the intermediate components:
A=>x=>B
Actually, I have determined that protein B is not controlled at the transcription level but post-translational level. So, I am believing in an unknown 'x' component in the middle which directly control the level of the protein B.
Regards
Can you determine what the posttranslational modification is? Either by mass spec, or trying various inhibitors of kinases, ubiquitination, unfolded protein response, etc. to see how your favorite protein reacts.
Thank you Ms. Jennifer for your reply...
Yeah... Immunoprecipitation in tendem with mass-spec is an option...
Thanks again...
Hi Mazhar,
here a couple road to do this:
1) Genetic: You could dissect the pathway by genetic. If the assumption is that A regulate B stability through x factor, I would make a reporter vector where B is fused to GFP and test removal of factors one-by-one in deletion/temperature sensitive strains (easily achieve in yeast-hopefully there are homolog to your factors) or in RNAi strains.
(here some example PMID: 17346711, PMID: 20349460)
2) Physical interaction: You could determine physical interactors of B and test if they are responsible for its stability. This can be achieve by immunoprecipitation follow by mass spectrometry. However for transient interaction, such as E2, E3 ligase, I would try an in vivo reporter such as BRET, FRET, Protein-fragment compleentation assay (PCA).
(here some example PMID: 17675294, PMID: 24019491)
3) Computational inferences: You could determine protein that share characteristic such as co-expression, similarity in evolution rates, complementary interaction domain (ipfam), etc to narrow down the potential regulators. Program such as STRING can built network in various organism based on these criterions.
Good luck ;)
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